This invention relates to a method and apparatus for the analyzation and sorting of particles, e.g., in a flow cytometer. In the field of flow cytometry it is common to establish a stream of sheath fluid with a stream of particles suspended in that sheath fluid. This stream can then be perturbed such that droplets form and the particles are contained in the droplets as they break-off from the end of a contiguous stream. The droplets can then be sorted as desired by detecting desired particles and establishing a charge on an individual droplet just before it breaks away from the contiguous part of the stream. The droplet containing the desired particle can then be deflected with an electric field into a collection container. As part of this process, it is optimal to know when a droplet containing a desired particle reaches the charging location such that that particular droplet can be charged while droplets charging as few neighboring droplets as necessary. This allows the droplet containing the desired particle and possibly a few droplets on either side of the droplet containing the particlexe2x80x94to be deflected into a separate container and sorted out of the stream.
As part of this process it has been necessary to set up a flow cytometer on a daily basis and to allow that flow cytometer to equilibrate to the environmental conditions where the flow cytometer is located. This takes approximately an hour to an hour and a half just for the process of equilibrating the flow cytometer. Then, typically another one-half hour is required to calibrate the drop delay timing of the flow cytometer after the equilibration period expires. Therefore, a full one to two hours is required on a daily basis just for setup of the flow cytometer. This is time that could be used for producing results from the flow cytometer rather than wasting it on setup time. Therefore, there is a desire for a flow cytometer that does not require this one to two hour setup time and that can be implemented quickly without the need for equilibration and calibration.
Another drawback to the present state of flow cytometry is the lack of an automatic means of compensating for change in one of the parameters of the flow cytometerxe2x80x94most importantly, the drop delay time. For example, it is currently necessary for a technician to monitor a sorting flow cytometer during the process of sorting. The technician must remain in the room while the sort is being performed in case a catastrophic failure of the flow cytometer would occur. In such a case, the technician could then, as quickly as possible, interrupt the sort and prevent any gathered sample of cells, for example, from being contaminated during a catastrophic failure. This might occur, for example, if a nozzle becomes clogged and the stream is angled away from the nozzle tip toward one of the sample collectors. Even with a technician in the room watching the sort take place, it would still require possibly two to three seconds for the flow cytometer to be stopped. In the case of some types of sorts, however, even this two to three second period would be too long to save the sort. Therefore, the process would have to be re-started and performed again. This can be quite frustratingxe2x80x94particularly if the sort had been near completion.
Furthermore, currently no warning system appears to exist when a parameter of the flow cytometer is set up in an incorrect manner. For example, if an incorrect nozzle size has been put on the flow cytometer, no manufacturer appears to be issuing a warning that can be used to alert the technician that the wrong nozzle size is attached. Therefore, this can result in unnecessary time on the part of the technician in trying to determine the problem with the setup of the flow cytometer.
Another drawback to the present state of the art in flow cytometry is the inability to determine a drop delay time for a particle to the degree of precision desired. Presently, one method that is used is to establish the stream and strobe the stream with a light source such that the stream can then be viewed on a monitor to see if the droplet break-off point of the stream changes position. If the break-off point shifts, then the stream can be re-calibrated to set the drop delay time for a particle. This is deceptive however, because a change in wavelength of the stream might occur without a resulting change in the droplet break-off point. Consequently, the drop delay time for a particle would changexe2x80x94as the change in wavelength would indicate a change in speed of the fluid flow. However, this would go unnoticed by a technician who was relying on the droplet break-off point position. It also assumes that the hydrodynamics of the stream are constant once the stream leaves the nozzle of the flow cytometer and thus, assumes the velocity of the stream remains constant.
Prior work in the field of flow cytometry apparently has been unsuccessful in solving these problems. Furthermore, they have focused on maintaining the droplet break off pointxe2x80x94rather than appreciating the ability to determine a drop delay time for a particle. For example, U.S. Pat. No. 4,691,829 to Robert E. Auer tried to utilize a laser beam aimed at the stream above the droplet break-off point. Based on refractive properties of the stream, it was then attempted to detect changes in the surface of the stream. A change in the undulations of the surface could then be used to determine when the break-off point had shifted. However, this method did not actually determine a drop delay time for a particle detected in the stream. It merely tried to maintain the droplet break-off point at the same position. Furthermore, it required very sensitive equipment to detect the change in the undulation of the surface and has apparently since the patent issued in 1987 never been made to work in a commercial product.
An earlier attempt to try to control the droplet break-off point can be seen in U.S. Pat. No. 3,761,941. In that patent, a test sample was run through the cytometer to try to detect a charge on a droplet. A theoretical charge that was expected to have been applied to the droplet was then compared to the actual charge on the droplet. The amplitude of the drop stimulating disturbance was then adjusted until the actual charge approached the theoretical charge. In this manner, the stream could be adjusted to the correct point for charging purposes.
In 1982, U.S. Pat. No. 4,361,400 discussed the use of a television monitor to view the breakoff point of a cytometer. However, it also required the operator to manually adjust the settings of the cytometer based on the viewed breakoff point. Therefore, equilibration of the cytometer was still likely a one and a half hour procedure if this method were used.
In 1997, U.S. Pat. No. 5,700,692 discussed the use of a camera/monitor system to allow a user to adjust the distance between droplets in a cytometer. However, it did not appreciate the ability of a monitoring system to determine a wealth of other characteristics of a stream and thereby automatically provide feedback to the flow cytometer. Instead, it focused on determining a center of mass of droplets and assumed a constant velocity of the fluid stream. In focusing on the center of mass of droplets, it apparently completely overlooked important information that could be determined from the streamxe2x80x94including an automatic regulation of a drop delay time.
Consequently, there is still a need for a flow cytometer that can monitor a stream of the cytometer and detect a drop delay time based on the specific characteristics of the stream at a specific point in time. Rather than relying on an expected steady state condition, such as a constant velocity of the stream, there is a need for a cytometer that can determine the drop delay time under the specific conditions of the stream for a particular particle that is about to be sorted. Furthermore, there is a need for a flow cytometer that can adjust the drop delay time at the beginning of the day when the flow cytometer is still adjusting to environmental conditions such as room temperature. In this way, the flow cytometer can be used for useful sorts during the first one to two hours that were previously required for equilibration to environmental conditions and calibration of the flow cytometer, such as calibration of the drop delay time using a standard test sample. There is also still a need for a flow cytometer that can detect when a catastrophic event occurs that could result in the destruction of a nearly completed sortxe2x80x94for example a five to six hour sort that is contaminated when a nozzle becomes blocked and the stream is inadvertently diverted into the sample collection container. In addition, there is still a need for an automatic interrupter that can divert or block a stream or turn off the sorting aspect of the stream automatically upon the occurrence of an event such as a catastrophic failure. In this manner the collected sample could be protected in as fast a time as possible, especially faster than the two to three seconds that would be required if an operator were to do it by handxe2x80x94as is apparently the case with current cytometers.
In addition, there is a need to understand the characteristics of the speed of the stream that is ejected by a flow cytometerxe2x80x94especially from the time that the stream is ejected from the flow cytometer through the point where a droplet is charged so that a charge can be applied at the droplet when the droplet reaches the charging location. In the past, it has been assumed that the speed was constant. However, as throughput is increased and particles become closer to one another in the stream, it is even more critical to be able to determine the speed of the stream drop delay time as accurately as possible; therefore, it is equally critical to understand the characteristics of the stream rather than simply estimate the stream as having a constant velocity.
The present invention provides a novel method of compensating for changes in a flow cytometer and accurately determining a drop delay time for a particular particle in the stream. A detected droplet can be charged based on a drop delay time that is computed based on a measurement of the speed of the fluid in the stream. Therefore, knowledge of the droplet charging location and the speed characteristics of the stream allows the cytometer to more accurately charge the droplet containing the particle at the droplet charging location.
In addition, other embodiments of the invention allow an image of the stream to be captured to determine information about the flow cytometer. For example, an image of the stream can be used to determine the width of the stream at a given point and correlate this to a nozzle size being used by the cytometer. In this fashion, it can be determined whether the correct nozzle size is being used. Other parameters can be determined in this fashion as well. For example, a velocity of the stream can be determined at various points along the stream. A velocity of the stream can be determined by measuring a wavelength of a surface wave on the stream and knowing the oscillation frequency that is being used by the mechanical device perturbing the stream in order to calculate the velocity of the stream at that point. Or, droplets occurring below the droplet break-off point can be imaged and a distance between the two droplets determined in order to determine the speed of the stream at the droplet break-off point.
Furthermore, an exponentially decaying model can be used to model the change in velocity of the stream below the nozzle exit pointxe2x80x94particularly between the nozzle exit point and the droplet break-off point for the stream fluid. This model can then be used to determine a more exact drop delay time for a particle detected in the stream.
In addition, the image monitoring system can be used to image the droplet break-off point to determine a location of the droplet break-off point, as well as a change in position of the droplet break-off point. Furthermore, the system can be used to provide a feedback signal to the flow cytometer such that the droplet break-off point is re-established or maintained at the desired position.
Also, the image monitoring system can be used to determine the effect of a charged droplet on a successive droplet or droplets. This can be accomplished by monitoring the trajectory of a droplet in real time and charging the subsequent droplet with a slight charge that allows it to fall in line with other droplets that do not contain particles.
The imaging system can also be used in a similar manner to determine the most stable position of the stream for a given pressure and oscillation. In this manner the preferred resonant frequency for a stream can be selected and the resulting stream established in a stable position that will not require oscillating between positions.
The invention can also be used to warn an operator of the flow cytometer that an anomaly exists in the setup or operation of the flow cytometer. In this fashion, the cytometer can perform a self-test during setup as well as during operation. For example, a determination of the speed of the stream could be used to determine whether the correct hydraulic pressure is being used for the flow cytometer. Or, a warning can be issued to the operator inquiring whether a different nozzle size is intended based on a width of the stream, for example. Similarly, a shift in position of the stream could be used to determine if the nozzle has become clogged or whether some other anomaly exists with the cytometer. Furthermore, if the stream is detected to have disappeared completely, the cytometer could warn the operator that a catastrophic event that would damage the sort had occurred. These warnings might be issued to either a remote monitor, a paging device, alarm device, or even an e-mail message.
Furthermore, rather than simply issuing a warning to the operator, mechanical intervention can be utilized to automatically or manually divert the stream and prevent it from contaminating a gathered sample. This might be accomplished using either a gutter or deflector. Alternatively or concurrently, one embodiment of the invention allows the sort to be disabled, either manually or automatically, by disabling the charging of the stream and/or disabling the deflection force that typically deflects a charged particle.
The invention also utilizes imaging methods to remove electrical noise from the captured images. For example an image of the stream can be captured and outlined to establish a first background image of the stream and then used as a template for comparison of subsequent images of the stream. In this fashion, electrical noise can be removed on subsequent images and the outline of the images compared to see if there has been a change in a characteristic of the stream.